A PCR-based test suitable for screening for fragile X syndrome among mentally retarded males

Abstract

Ever since the identification of the genetic cause of fragile X syndrome as the expansion of an unstable trinucleotide sequence, several diagnostic strategies have evolved from molecular studies. However, we still lack a simple test suitable for population screening. We have therefore developed a nonisotopic polymerase chain reaction (PCR)-based technique for the identification of fragile X full mutations among men, with easy visualization of the PCR products on silver-stained polyacrylamide gels. The technique consists of PCR amplification with primers that flank the trinucleotide repeats, with a product of 557 bp for the (CGG)29 allele. Conditions were established such that full mutations failed to amplify and were thus identified with 98% sensitivity compared with Southern blot analysis. To produce an indispensable internal control we added to the reaction a third primer, internal to this fragment, allowing the multiplex amplification of a monomorphic band corresponding to a CG-rich stretch 147 bp upstream of the polymorphic region. In trials involving 41 patients and 74 controls, the PCR-based test here described showed specificity of more than 98.6%, accuracy of 99% and a sensitivity of 98%. Thus, although not suitable for medical diagnosis, it constitutes a useful tool for screening for the fragile X syndrome in populations of mentally retarded males.

Association between the oxytocin receptor (OXTR) gene and autism: relationship to Vineland Adaptive Behavior Scales and cognition

“…All subjects tested negatively for Fragile-X using a PCR procedure with an ABI 310 DNA analyzer. [28][29][30] In addition, none of the subjects was known (according to parents' reports) to have any other chromosomal abnormality or tuberous sclerosis.…”

Short Communication Screening for fragile X syndrome in males from specialized institutions in the northeast region of Brazil

“…Screening for FMR1 gene mutations was performed using the non-isotopic polymerase chain reaction (PCR) method described by Haddad et al (1996). The primers used to amplify the 223-bp region (monomorphic control localized next to trinucleotide CGG repetitions in the 5'-untranslated region of the gene) and the polymorphic fragment of the CGG repeat are shown in Table 2.…”

“…For evaluation of the electropherogram generated by the sequencing system we used the program Chromas Lite Version2.0 (Technelysium, South Brisbane, Australia) and for alignment of the patient sequences with their respective wildtype sequences we used the program Bioedit Sequence Alignment Editor Version 7.0.5.2 (Isis Pharmaceuticals, Inc., Carlsbad, CA, USA). Primers and PCR conditions are presented as reported in Haddad et al, 1996. PCR = polymerase chain reaction.…”

Clinical checklists in the selection of mentally retarded males for molecular screening of fragile X syndrome

“…DNA samples were analyzed using the PCR according to Haddad et al (1996). In this method genomic DNA is amplified by one forward and two reverse primers in two combinations, one pair of primers flanking the trinucleotide repeat region amplifies a fragment containing the variable CGG region while the other pair of primers amplifies a smaller 223 bp fragment of the CGG region used as an internal control.…”